ORIGINAL ARTICLE

Effect of carotenoid β-cryptoxanthin on cellular and humoralimmune response in rabbit

Soroush Ghodratizadeh & Güngör Kanbak &

Mojtaba Beyramzadeh & Zeliha Gunnur Dikmen &

Salar Memarzadeh & Reza Habibian

Accepted: 6 November 2013 /Published online: 5 December 2013# Springer Science+Business Media Dordrecht 2013

Abstract Beta-cryptoxanthin (b-Cr) is a pro-vitamin A andone of the major carotenoids that can be commonly found inmammalian serum and tissues. Foods rich in certain fatty acidsare known to be effective to gain a healthy immune system. Inthe present study, we evaluated the effect of b-Cr on rabbithumoral and cellular immune responses to have a better visionabout the mechanism of effect of carotenoids on immunesystem. Twenty rabbits were randomly divided into fivegroups (4 per group): Groups consisted of: 1) control group(normal saline; 2) b-Cr (control); 3) vaccine control; 4)5 mg/kg b-Cr o.p. + vaccine; 5) 10 mg/kg b-Cr o.p. + vaccine.Blood samples were obtained from the marginal ear artery atthree time points: days 0, 14 and 21 of the study. BloodCD4+ and CD8+ lymphocytes and Serum Immunoglobulinand Cytokines content were evaluated. Results show that b-Cradministration increased the blood CD4+ lymphocytescount (P >0.01). Serum IgG, IgM and IgA levels increased(P >0.05) following b-Cr administration. b-Cr treatment in-creased serum IL-4 levels (P >0.05). According to presentedresults, b-Cr may increase the humoral immunity in mammals.So, it would possible has a potentially beneficial effect onhealth and on prevention of the immunity related diseases.

Keywords β-cryptoxanthin . Serum Immunoglobulin .

Cytokine . CD4+ cells

Introduction

Carotenoids are a family of coloured tetra-terpenoids, which arehydrophobic and totally have a symmetric or quasi-symmetricstructure. Some of carotenoids are pro-vitamin A, which isoxidatively cleaved to vitamin A by b-carotene- 15,150-monooxygenase (Wyss A 2004). Vegetables and fruits are richin carotenoids, which protect them from oxidative stress. Beta-cryptoxanthin (b-Cr), related to b-carotene in terms of the struc-turewith the addition of a hydroxyl group, is a pro-vitaminA andone of six major carotenoids (b-carotene, lycopene, lutein, b-Cr,zeaxanthin, and a-carotene) that can be commonly found inhuman serum and tissues (Khachik F et al. 1997). Like othercarotenoids, b-Cr has an antioxidant and free radical scavengereffect (Miller NJ et al. 1996) and stimulates the repair of oxida-tive damage toDNA (LorenzoYet al. 2009) suggesting that b-Crhas a wide range of beneficial effects in the orgasm body.

Generally, the immune system function, like other systemsin the mammalians body, is dependent on proper nutrition. Ithas been well known that overnutritional condition and severemalnutrition lead to immunodeficiency, resulting in infectionsand health problems (Chandra RK 1997). Malnutrition is themost common cause of immunodeficiency, suggesting thatwell balanced diet may be highly important for retaining orimproving the immune function in animals. Fresh vegetablesand fruits, foods rich in certain fatty acids, are knownto be effective to gain a healthy immune system while pro-inflammatory fatty acids cause an imbalance in the immunefunction (Pond CM2005 ). In herbal medicine, some herbs arebelieved to stimulate the immune system (Spelman K et al.2006; Clark CE et al. 2010). Carotenoids may be the maintherapeutic contents of these herbs. In the present study, we

S. Ghodratizadeh :G. KanbakFaculty of Medicine, Department of Biochemistry, OsmangaziUniversity, Eskisehir, Turkey

M. Beyramzadeh (*) : Z. G. DikmenDepartment of Biochemistry, Faculty of Medicine, HacettepeUniversity, Ankara, Turkeye-mail: mojtaba@hacettepe.edu.tr

S. MemarzadehFaculty of Medicine, Urmia University of Medical Sciences, Urmia,Iran

R. HabibianDivision of Immunology, Department of Microbiology, Faculty ofVeterinary Medicine, Urmia University, Urmia, Irane-mail: r.habibian@urmia.ac.ir

Vet Res Commun (2014) 38:59–62DOI 10.1007/s11259-013-9584-8

evaluated the effect of b-Cr on rabbit humoral and cellularimmune responses to have a better vision about the mechanismof effect of carotenoids on immune system.

Material and methods

Animal design

In this study 20 male New Zealand white rabbits, weighing2.2 to 2.6 kg, were used. The rabbits were kept for 10 days toadapt to the environmental conditions. They were housed inindividual cages in a room with controlled temperatureof 20±2 °C, and a 12 h light/dark cycle. The rabbits were fedstandard diet and had access to fresh water ad libitum. Animalswere randomly divided into five groups (4 per group): Groupsconsisted of: 1) control group (normal saline, o.p.); 2) b-Cr(97 % pure, Sigma, US) o.p. (control); 3) vaccine control; 4)5 mg/kg b-Cr o.p. + vaccine; 5) 10 mg/kg b-Cr o.p. + vaccine.According to the literature (Nishi et al. 2012), the minimumeffective doses of b-Cr in lab animal studies are 2–10 mg/kg.The b-Cr was dissolved in corn oil at a concentration of 5 or10 mg/ml and then 5 or 10 mg/1 ml/kg body weight was orallyadministered to rabbits through a syringe once daily for 21 days.Control animal received corn oil (1 ml/kg body weight) orally.Vaccination was done on day 0 of study. All animal experimentsdescribed in this paper, were carried out in accordance withprotocols approved by the hacettepe University Animal Careand Use Committee and were performed in accordance withapplicable guidelines and regulations for the Care and Use ofLaboratory Animals of hacettepe University. The rabbit vacci-nated by myxomatosis vaccine (Mixohipra-FSA®, LaboratoriosHipra, S.A. one dose/rabbit). Blood samples were obtained fromthe marginal ear artery using 25 G catheter at three time points:days 0, 14 and 21 of the study. Blood samples were transferredinto heparinised and none heparinised tubes. For immunologicalanalysis, the serum was separated by centrifugation at 3,000 rpm/min for 10 min and stored at −20 °C until analysis.

Immunoglobulin and cytokine content

Serum immunglobulin and cytokine concentrations weremea-sured by ELISA technique using commercial ELISA test kit

for IgA, IgG and IgM (GenWay Biotech Inc. San Diego, CA)and interleukin-4 (IL-4) and interferon (IFN) gamma (genxbioInc. delhi, India) according to product description.

Flow cytometric analysis

Peripheral blood mononuclear cells (PBMCs) were isolatedby standard Ficoll density gradient centrifugation (Nieto JCet al. 2012). Monoclonal antibodies and negative controlsused to quantitate the rabbit lymphocyte subsets werepurchased from Serotec (Oxford, UK). The antibodies(AbD Serotec, Oxford, UK) were specific for the followinglymphocyte subsets: anti CD4 and anti CD8 lymphocytes.Appropriate negative controls were included to correct back-ground fluorescence. Data were expressed as the percentageof positive staining cells corrected for cells stained nonespecifically with the secondary antibody. The cells were ana-lyzed by flow cytometry with a BD-FACSCalibur flowcytometer (BD Biosciences, San Jose, CA). The data wereanalyzed using BD CellQuest Pro software. About 20,000lymphocytes were examined for each sample.

Statistical analysis

Results were expressed as mean ± S.E.M. Multiple compari-sons were performed by ANOVA followed by the Tukeyhonestly significant difference (HSD) test. In all the analyses,the level of significance was set at P <0.05.

Results

Effect of b-Cr on CD4+ and CD8+ lymphocytes

Table 1 shows the effect of b-Cr on the CD4+ and CD8+lymphocytes in the blood of rabbits in different groups.Vaccine administration increased CD4+ cells as expected.Administration of 10 mg/kg b-Cr produced a significant(P <0.05) increase in the blood CD4+ lymphocytes after 14and 21 days, when compared with the control group. Therewas no significant (P >0.05) change in CD8+ lymphocytesfollowing b-Cr treatment.

Table 1 Effect of b-Cr on CD4+and CD8+ lymphocytes. Thevalues are presented as mean ±S.E.M

a, b: Data with different letters(a,b) in the same column are sig-nificantly different at P<0.05

CD Day Group 1 Group 2 Group 3 Group 4 Group 5

CD4+(%) 0 41.4±3.4 40.4±3.1 39.6±3.5 42.2±4.2 41.2±3.2

14 40.1±4.3 42.5±4.2 45.4±4.3a 46.8±4.7a 55.4±4.3b

21 39.8±4.5 41.2±4.9 44.7±.3.6a 45.2±4.6a 54.7±4.6b

CD8+(%) 0 19.3±1.8 20.4±1.9 18.7±1.7 19.2±2.1 20.8±1.9

14 18.8±1.7 19.4±1.7b 18.2±2.3 19.4±2.5 18.7±2.5

21 19.1±2.0 20.7±1.8a 19.5±2.4 20.2±2.3 19.4±2.8

60 Vet Res Commun (2014) 38:59–62

Effect of b-Cr on serum IgM, IgG and IgA levels

According to Table 2, serum IgM concentrations in both dosesof b-Cr increased significantly (P <0.05) on days 14 and 21.Serum IgG concentrations in group 4 increased significantlyonly on day 21 (P <0.05), whereas its levels in group 5increased both on days 14 and 21 (P <0.05). Serum IgAconcentrations in group 4 and 5 increased significantly onlyon day 21 (P <0.05). According to presented results, b-Cr hasdifferent effects on immunoglobulins production in a dosedependent manner. In addition, Vaccine administration slight-ly increased IgM and IgG levels.

Effect of b-Cr on serum IL-4 and IFN-gamma levels

According to Table 3, serum IL-4 concentrations in groups 4increased significantly (P <0.05) on day 21. On the otherhand serum IL-4 concentrations in group 5 increasedsignificantly (P <0.05) on days 14 and 21 when comparedto the control. Serum IFN-gamma concentrations did notshow any significant difference (P >0.05) following b-Crtreatment. Vaccine control group show a mild increase inIL-4 and IFN-gamma levels.

Discussion

The effects of carotenoides on immune system are well doc-umented (Chew BP and Park JS 2004). However there is

limited knowledge about its details. There are various indexesto evaluate the effect of biochemical compounds on functionof the immune system. For example, the composition oflymphocyte subsets, different cytokines levels and serumimmunoglobulins are employed for the evaluation of the cellmediated or humoral immune responses. A CD4+ count is ablood test to determine how well the immune system isworking and is known as humoral immune response indicator(Shirwan H et al. 2003). CD8 is a trans membrane glycopro-tein that acts as a co-receptor for the Tcell receptor (TCR). It ispredominantly expressed on the surface of cytotoxic T cells,so is an indicator for cellular immune response (Vecchione Aet al. 2002).

According to results of present study, administration of b-Cr can change the immunological factors in a positive way.Results show that b-Cr increased the IgM levels dose inde-pendently and CD4+ lymphocytes count, IgG, IgA and IL-4levels dose dependently.

Okai and Higashi-Okai (1996) and Nishi K et al. (2012)reported that treatment of carotenoids increases production ofIgG and IgM by splenocytes fromBALB/cmice in vitro. So, itseems that substances in b-Cr have Ig production-stimulatingactivity. Chew et al. (2000) reported that an increase in con-centration of IgG, but not IgM, was found in serum of the b-carotene administered dogs than in none administered dogs.The result in this paper is similar to our founding in productionof IgG and IgM, implying that the beta-carotene administra-tion might have an effect on the immunoglobulin productions.However our results show the same effect on IgA, too.

Table 2 Effect of b-Cr on serumimmunoglobulins (IgG, IgM, andIgA) concentration. The valuesare presented as mean±S.E.M

a, b: Data with different letters(a,b) in the same column are sig-nificantly different at P<0.05

Immunoglobulin Day Group 1 Group 2 Group 3 Group 4 Group 5

IgG (mg/ml) 0 7.1±0.34 7.2±0.31 6.9±0.35 7.2±0.42 7.3±0.32

14 7.2±0.43 7.0±0.42 8.4±0.43a 8.8±0.47a 9.4±0.43b

21 7.8±0.45 7.2±0.49 8.7±0.36a 8.8±0.46a 9.7±0.46b

IgM (ng/ml) 0 8.3±0.68 7.7±0.79 8.1±0.77 7.9±0.61 8.3±0.39

14 8.5±0.67 7.9±0.37 8.9±0.63a 9.8±0.85b 9.7±0.54b

21 8.3±0.50 7.7±0.48 9.1±0.94a 10.5±0.83b 11.2±0.78b

IgA (ng/ml) 0 6.3±0.68 6.4±0.19 6.7±0.47 6.2±0.61 6.8±0.59

14 6.8±0.37 6.4±0.17 6.2±0.23 6.4±0.65 6.7±0.85

21 6.3±0.20 6.7±0.48a 6.5±0.44 7.5±0.23a 8.6±0.38b

Table 3 Effect of b-Cr on serumcytokines (IL-4 and IFN-gamma)concentration. The values arepresented as mean±S.E.M

a, b: Data with different letters(a,b) in the same column are sig-nificantly different at P<0.05

Cytokine Day Group 1 Group 2 Group 3 Group 4 Group 5

IL-4(pg/ml) 0 4.4±3.4 4.4±3.1 3.9±3.5 4.2±4.2 4.0±3.2

14 4.1±4.3 4.5±4.2 5.4±4.3a 5.8±4.7a 7.4±4.3b

21 3.9±4.5 4.2±4.9 5.5±.3.6a 6.9±4.6ab 8.2±4.6b

IFN-gamma (pg/ml) 0 8.3±1.8 8.4±1.9 8.7±1.7 8.2±2.1 8.8±1.9

14 8.6±0.72 8.4±0.67 9.6±0.33a 9.7±0.55a 9.7±0.25a

21 8.3±0.52 8.2±0.38 9.5±0.44a 9.5±0.43a 9.4±0.28a

Vet Res Commun (2014) 38:59–62 61

Effects of b-Cr on serum cytokines content were also exam-ined. Statistically significant differences between b-Cr treatedand control groups were observed in IL-4 production with adose-dependency. IL-4 level increased in 10 mg/kg dose group,whereas no change observed in 5 mg/kg dose group. IL-4 is aTh2 cells product and humoral immune response mediator. IL-4levels support the result of CD4+ count. Productions of IFN-gamma not changed significantly. IFN-gamma is Th1 cellsproduct and cellular immune response mediator. IFN-gammaconcentration was in accordance with CD8+ cells count. Nishiet al. (2012) reported b-Cr administration cause an increase inIL-6 levels but did not affect the IL-4 and IFN-gamma levels.These results might be related to the significant increase inimmunoglobulin levels and CD4+ cells count in the b-Cr treatedgroups. It has been reported that oxygenated carotenoidslike b-Cr in some vegetables and fruits are esterifiedwith different fatty acid chains (Dhuique-Mayer C et al.2007). This esterified form of xanthophyll has a higher bio-availability than its free form counterpart (Fu et al., 2010). So,esterified b-Cr with a fatty acid chain could be useful to furtherexamine the immunostimulatory effect of b-Cr.

On the other hand, the functional effect of a carotenoid isdetermined by its sub cellular localization. Studies in cats (ParkJS et al. 1999) and dogs (Chew BP et al. 1998) have shownsignificant uptake of orally fed lutein by the mitochondria,microsomes and nuclei of lymphocytes. Similar studies in cats(Chew BP et al. 1998), dogs (Chew BP et al. 2000), reporteduptake of beta-carotene by all lymphocyte sub cellular fractions.

Themitochondria electron transport system utilizes more than85 % of the oxygen consumed by the cell to generate ATP;therefore, they are the most important source of reactive oxygenspecies (Shigenaga MK et al. 1994). Cytochrome c locatedbetween the inner and outer mitochondria membranes plays acritical role in the apoptotic process. The release of cytochrome cis regulated by the pro-apoptotic proteins BAX, BID and BIM,and by the anti-apoptotic proteins Bcl-2, Bcl-XL andBFL-1. Therefore, the mitochondria could be a key player inimmunity and disease, and the localization of the carotenoidsin the mitochondria is therefore of particular relevance. Thepresence of these carotenoids in sub cellular organelles canprotect the immune cells against oxidative injury, and ensureoptimal cellular functions, including cell signalling, gene reg-ulation and apoptosis (Chew BP and Park JS 2004).

Conclusion

B-Cr shows a stimulating activity on immunoglobulin pro-duction, Th2 cytokines and CD4+ cells in rabbit. According topresented results, b-Cr may increase the humoral immunity inmammals. So, it would possible has a potentially beneficialeffect on human health and on prevention of the immunityrelated diseases.

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